CHARACTERIZATION OF THE PROMOTER OF THE RAT SARCOPLASMIC ENDOPLASMIC-RETICULUM CA2-ATPASE-1 GENE AND ANALYSIS OF THYROID-HORMONE RESPONSIVENESS()

Relaxation of skeletal muscle requires the re-uptake of Ca2+, which is mediated by the sarcoplasmic reticulum Ca2+-ATPase (SERCA). Thyroid h ormone (T-3) stimulates the expression of the SERCA1 isoform, which is essential for fast skeletal muscle fiber phenotype. We have cloned an d studied the first 962 base pairs of the 5'-flanking region of the ra t SERCA1 gene. This sequence was tested for T-3-regulated expression i n transient transfection experiments using COS7 cells and for binding of thyroid hormone receptor (TR) alpha in mobility shift assays. A con struct of the 5'-flanking region and a reporter gene was unresponsive to T-3 in the absence of co-transfected thyroid hormone receptor. In t he presence of TR alpha T-3 induction ratio of almost 4.0 was found, a nd this induction ratio was doubled with co-transfection of an RXR exp ression plasmid. Analysis of progressive 5'-deletion fragments of the sequence indicated multiple regions involved in T-3 responsiveness. Th ree regions, R1, R2, and R3, were identified that bound TR complexes i n mobility shift assays and conferred T-3 responsiveness to a heterolo gous promoter. The most potent of these thyroid hormone response eleme nts, R3, increased the 2-fold background T-3 stimulation of the thymid ine kinase promoter to nearly 6-fold. Detailed analysis of this elemen t showed that four TR-binding half-sites, comprising two independent t hyroid hormone response elements, interact cooperatively to give the m aximal T-3 response. T-3 regulation of SERCA1 expression is mediated b y a complex thyroid hormone response element that may serve to provide a greater range of response in interaction with nuclear receptor part ners or cell-specific transcription factors.